Molecular cloning, characterization and overexpression of a novel cyclin from Leishmania mexicana

We are reporting here, the cloning and characterization of the first cyclin from Leishmania mexicana. We have identified a cyclin-like motif from the L. major genome sequencing project. A cyclin homologue was cloned and sequenced from L. mexicana genome and it showed 96.1% amino acid identity with the putative L. major cyclin. It has also sequence identity to mitotic cyclins from other organisms. Southern analysis showed that it is present as a single copy gene. CYCa has been over-expressed in E. coli as a histidine fusion and western blot has confirmed the immunoreactive property of the recombinant cyclin, which then used to reconstitute active recombinant L. mexicana CRK3. No phosphorylation of histone HI was detected by both wild type and mutated CRK3 on the activation assays suggesting that phosphorylation status and cyclin binding are important for reconstituting protein kinase activity. The results confirm that we have isolated a cyclin molecule from L. mexicana (LmCYCa) which may play an important role in the regulation of the parasite cell cycle.     

花生果种皮特异表达基因AhPSG13的克隆和表达研究

为了克隆在花生果种皮中特异表达的基因,本实验从花生果皮种仁抑制消减文库中筛选出一个277bp的EST序列并进行研究,通过构建花生果皮全长cDNA文库,获得此目的基因G13的全长为1369bp,开放阅读框从第28个碱基始至1122个碱基止,预测分子量为39865.59,等电点5.73。生物信息学分析表明该基因编码的蛋白具有4个跨膜结构域和多个活性位点,可能与细胞内DNA转录有关;该蛋白与多种豆科作物的半胱氨酸蛋白酶具有较高同源性,推测为花生半胱氨酸蛋白酶相关基因;RT-PCR研究该基因表达,结果显示该基因在果种皮中特异表达,于30d果皮中表达量最大。本研究克隆的AhPSG13基因序列已经登陆到GenBank,登录号为FJ475061。     

Molecular cloning and expression analysis of the main gliadin-degrading cysteine endopeptidase EP8 from triticale

During the development and maturation of cereal grains, storage proteins are accumulated in the starchy endosperm. The enzymes responsible for the mobilisation of stored proteins during seed germination are cysteine endopeptidases and serine carboxypeptidases. In our previous study, we purified the major gliadin-degrading cysteine, endopeptidase EP8, from germinating triticale kernels. We confirmed in an experiment with exogenous gibberellic acid (GA₃) that this enzyme is synthesised in aleurone during germination. In this study, the nucleotide sequence of EP8, a barley EP-A (HvEPA) homologue, and the expression pattern during grain development and germination have been analysed. Additionally, by comparing the activity of purified EP8 with the activity pattern of crude enzymatic extracts, the lack of enzyme activity in some tissues and during certain developmental stages has been demonstrated. The correlation of the results of the EP8 expression analysis with the activity pattern of this endopeptidase allowed us to formulate a hypothesis regarding the mechanism of EP8 activity regulation. We believe that the absence of active EP8 until the third day of germination may be associated with high levels of endogenous cystatin TrcC-4, which has the ability to inhibit EP8 in vitro.     

大豆中GmKAB1基因的克隆和信息学分析

为探讨GmKAB1在大豆处于低钾胁迫下的表达水平,以大豆钾高效品系油06-71和钾敏感型品系衡春04-11为试验材料,设置低钾胁迫试验,分别在处理后0.5h、2h、6h、12h、3d、6d、9d和12d取样提取RNA,利用Real time-PCR检测各时间段GmKAB1基因的表达量。结果显示GmKAB1基因在地下部分表达比地上部持续时间更长,地下部相对表达倍数最高为3.5,较地上部相对表达倍数更高。克隆目的基因并对基因序列进行同源性及生物信息学分析,结果表明,与GmKAB1基因相似性在30%以上的同源基因有45个,GmKAB1在进化树中的位置与Glyma20g19000最近;GmKAB1编码蛋白为稳定的可溶性蛋白,具有2个保守结构域,多个磷酸化位点,表明在受到低钾胁迫后该基因编码的β亚基与α亚基结合调控电压门控钾离子通道,对大豆从根部获取钾离子可能起着关键作用。     

马尾松银松素合酶基因启动子区的克隆及特征分析

银松素合酶（pinosylvin synthase,PS）是合成银松素类植物抗毒素的关键酶。本文根据前期工作所获得的PS基因序列,设计基因特异引物,利用连接介导的染色体步行技术,从马尾松总基因组中扩增PS基因上游的启动子区;通过PLAN-CARE等分析软件对5′侧翼区近700 bp进行启动子特征分析,预测启动子区调控元件。结果表明,在翻译起始密码子上游167 nt处可能存在TATA-box。此外还发现3个GATA-box（-260 nt、-295 nt、-386 nt和-295 nt）和若干个TGAC-like序列。     

甘蓝型油菜BnPex16p基因cDNA的克隆与表达

利用模式植物拟南芥AtPex16p/sse1基因序列与油菜数据库BBSRC BrassicaDB比对,得到甘蓝型油菜(Brassica napus)同源EST序列,拼接出油菜Pex16p基因全长cDNA电子克隆,然后设计全长引物,以甘蓝型油菜种子cDNA为模板,克隆Pex16p基因,得到全长为1 101bp的cDNA,编码366个氨基酸的蛋白,命名为BnPex16p。BnPex16p基因序列与拟南芥AtPex16p/sse1同源,其蛋白与拟南芥同源蛋白具有完全相同的PTS2型过氧化物酶体定位肽,进化关系相近。半定量PCR(RT-PCR)发现BnPex16p基因在油菜幼嫩的根、茎、叶和种子中均有高丰度表达,在种子发育过程的中期和后期表达水平增加,暗示该基因在油脂的积累中有重要作用。     

油菜乙酰乳酸合酶基因BnALS3的亚细胞定位、原核表达及其酶活分析

为了研究油菜乙酰乳酸合酶基因BnALS3的亚细胞定位与酶学特性,以甘蓝型油菜（Brassica napus L.）品系N131为材料,采用RT-PCR法克隆到BnALS3的cDNA序列。该序列含有一个长度为1 959bp的开放阅读框,编码蛋白为652个氨基酸,在N端含有一个由49个氨基酸残基组成的叶绿体信号肽序列。将该信号肽序列构建至植物表达载体35S:: BnALS3-GFP,利用拟南芥原生质体细胞进行瞬时表达,结果显示BnALS3蛋白定位在叶绿体中。构建去除叶绿体信号肽序列的BnALS3原核表达载体pCzn1-BnALS3,转入大肠杆菌BL21(DE3)进行诱导表达。SDS-PAGE和Western Blot检测显示,在11℃条件下以终浓度为0.2mmol.L-1的IPTG诱导8h后,pCzn1-BnALS3基因在大肠杆菌中成功表达,融合蛋白His-BnALS3呈部分可溶性,分子量约为67kD。酶活分析表明,原核表达的His-BnALS3最适反应pH值为7.0,最佳反应温度为37℃,酶学常数Km值和Vmax值分别为6.55 mmol·L-1和6.40μmol·mg-1·h-1。     

Molecular and Clinical Investigation of Iranian Patients with Friedreich Ataxia

Background: Friedreich ataxia (FRDA) is an autosomal recessive disorder caused by guanine-adenine-adenine (GAA) triplet expansions in the FXN gene. Its product, frataxin, which severely reduces in FRDA patients, leads to oxidative damage in mitochondria. The purpose of this study was to evaluate the triple nucleotide repeated expansions in Iranian FRDA patients and to elucidate distinguishable FRDA clinical differences in these patients. Methods: A number of 22 Iranian patients (8 females and 14 males) from 16 unrelated families were studied. DNA was extracted from the peripheral blood of patients. The frequency and length of (GAA)_n repeats in intron 1 of the FXN gene were analyzed using long-range PCR. In this study, the clinical criteria of FRDA in our patients and the variability in their clinical signs were also demonstrated. Results: An inverse relationship was observed between GAA repeat size and the age of onset. Although some distinguishable clinical features (such as limb ataxia and lower limb areflexia) were found in our patients, 90-95% of them had extensor plantar response and dysarthria. The results showed only one positive diabetes patient and also different effects on eye movement abnormality among our patients. Conclusion: The onset age of symptoms showed a significant inverse correlation with allele size in our patients (P>0.05). Based on comparisons of the clinical data of all patients, clinical presentation of FRDA in Iranian patients did not differ significantly from other FRDA patients previously reported. Key Words: Friedreich ataxia (FRDA), Frataxin, Mitochondria     

Molecular cloning and expression analysis of the Rf-m candidate genes encoding pentatricopeptide repeat proteins in soybean

Cytoplasmic male sterility (CMS)-restorer system is a useful tool to exploit heterosis in soybean. The major restorer gene for the M-type CMS is known as Rf-m, located in the 162.4-kb region on chromosome 16. Sequence analysis has revealed that the Rf-m locus in Glycine max consists of seven pentatricopeptide repeat (GmPPR) genes. The deduced amino acid sequences contain 8 to 14 PPR motifs, and a phylogenetic analysis grouped these GmPPR proteins into two PPR subfamilies: Glyma.16G161800 belongs to the PLS subfamily, and the P subfamily consists of Glyma.16G161900, Glyma.16G162000, Glyma.16G162100, Glyma.16G162700, Glyma.16G162800, and Glyma.16G163100. The phylogenetic analysis of seven GmPPR proteins and 27 other plant PPR proteins also showed that proteins in the same subfamilies cluster together. Comparative sequence analysis was conducted using the seven Rf-m candidate GmPPR genes from the sterile line W931A, the maintainer line W931B, and the restorer line WR016, the result showed that Glyma.16G161900 had higher polymorphism than the other candidate genes. Based on real-time quantitative RT-PCR data, all seven GmPPR genes were differentially expressed but showed constitutive expression in roots, stems, leaves, and pollen grains. Additionally, the expression level of Glyma.16G161900 in the sterile line W931A was significantly higher in all tissues than in the restorer line WR016. Taken together, these results suggest that Glyma.16G161900 is the most likely candidate for the restorer gene Rf-m. This study is the first report and analysis of candidate fertility restorer (Rf) genes encoding PPR proteins in soybean.     

芝麻主要过敏原油质蛋白的基因克隆和原核表达

为克隆黑、白芝麻主要过敏原油质蛋白Oleosins的基因并在原核细胞中对其进行表达,本研究分别提取黑、白芝麻的总RNA,逆转录合成cDNA,根据序列设计简并引物,扩增芝麻主要过敏原油质蛋白Oleosins基因的完整开放阅读框,并与pET-28a载体连接,测序鉴定后转化大肠杆菌E.coliBL21(DE3)及用IPTG诱导表达。结果表明克隆获得黑、白芝麻主要过敏原油质蛋白Oleosins的全长基因约为438bp的开放阅读框(包括终止密码子),编码145个氨基酸。所编码的蛋白相对分子质量约为15.5kDa,为小分子量蛋白,等电点为5.18,与理论值相符。序列同源性分析发现其与数据库中已知的黑、白芝麻油质蛋白Oleosins基因同源性很高(GenBank黑、白芝麻油质蛋白基因登录号分别为EU999158和EU999159),并在DE3中高效表达。     

Reporting Two Novel Mutations in Two Iranian Families with Cystic Fibrosis,Molecular and Bioinformatic Analysis

Background:Cystic fibrosis is the most common heredity disease among the Caucasian population. More than 350 known pathogenic variations in the CFTR gene ({"type":"entrez-nucleotide","attrs":{"text":"NM_000492.4","term_id":"1732746288","term_text":"NM_000492.4"}}NM_000492.4) cause CF. Herein, we report the outcome of our investigation in two unrelated Iranian families with CF patients. Methods:We conducted phenotypic examination, segregation, linkage analysis, and CFTR gene sequencing to define causative mutations. Results:We found two novel mutations in the present study. The first one was a deletion causing frameshift, c.299delT p.(Leu100Profs*7), and the second one was a missense mutation, c.1857G>T, at nucleotide binding domain 1 of the CFTR protein. Haplotype segregation data supported our new mutation findings. Conclusion:Findings of this study expand the spectrum of CFTR pathogenic variations and can improve prenatal diagnosis and genetic counseling for CF.Key Words: Cystic fibrosis, Cystic fibrosis transmembrane conductance regulator, Genetic linkage, Haplotype, Sequence analysis     

油菜PEPase基因的克隆及其对应RNAi载体的构建

磷酸烯醇式丙酮酸羧化酶(PEPase)是控制油菜蛋白质/油脂含量比例的一个关键酶.采用RT-PCR方法从甘蓝型油菜中双4号中扩增出PEPase基因cDNA片段,与已发表的PEPase基因(登录号为D13987)序列同源性为98%.根据RNA 干扰(RNAi)目标序列的选取原则选取两段长度约为400bp的DNA序列,分别克隆到RNAi载体pHBM1301中,构建了油菜中对应于PEPase基因的RNAi载体pHBM1301-PEP1和pHBM1301-PEP2.     

Effect of elatol, isolated from red seaweed Laurencia dendroidea, on Leishmania amazonensis

In the present study, we investigated the antileishmanial activity of sesquiterpene elatol, the major constituent of the Brazilian red seaweed Laurencia dendroidea (Hudson) J.V. Lamouroux, against L. amazonensis. Elatol after 72 h of treatment, showed an IC(50) of 4.0 μM and 0.45 μM for promastigote and intracellular amastigote forms of L. amazonensis, respectively. By scanning and transmission electron microscopy, parasites treated with elatol revealed notable changes compared with control cells, including: pronounced swelling of the mitochondrion; appearance of concentric membrane structures inside the organelle; destabilization of the plasma membrane; and formation of membrane structures, apparently an extension of the endoplasmic reticulum, which is suggestive of an autophagic process. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa, and it is not toxic to macrophages. Our studies indicated that elatol is a potent antiproliferative agent against promastigote and intracellular amastigote forms, and may have important advantages for the development of new anti-leishamanial chemotherapies.     

大豆天冬氨酸途径关键酶基因GmASADH的克隆与分析

本研究基于电子克隆获得的大豆GmASADH 基因cDNA序列,根据其编码区设计特异引物,以大豆品种小鹦哥豆总RNA为模板,通过RT-PCR 获得了约1130bp的cDNA片段,T/A 克隆后进行序列测定。测序结果显示,GmASADH基因家族有两个成员,命名为GmASADH1（FJ581425）和GmASADH2（HM015631）。GmASADH1编码区长度为1134bp,编码378个氨基酸,由7个外显子和6个内含子构成,定位于Gm08染色体。GmASADH2编码区长度为1131bp,编码377个氨基酸,由7个外显子和6个内含子构成,定位于Gm05染色体。两个基因在氨基酸水平上的相似性达到了96.02%,在二级及三级结构上均有不同程度的差异。ASADH蛋白含有NADB_Rossmann superfamily和Semialdhyde_dhC superfamily结构域。     

橡胶树β-淀粉酶基因HbBAM1的克隆与表达分析

叶绿体内的淀粉是植物光合作用产物的重要临时储存形式,这些淀粉的降解需要β-淀粉酶(BAM)。β-淀粉酶基因是一个多基因家族,分别在不同组织中起着不同的作用,叶绿体β-淀粉酶在叶绿体淀粉降解过程中起着关键作用。利用橡胶树的转录组和基因组数据库,通过RT-PCR的方法获得一个橡胶树BAM基因,命名为HbBAM1。利用实时荧光定量PCR分析其在叶片中的表达模式,通过原核表达获得其重组蛋白,并分析其酶活性特点。同源性分析和亚细胞预测分析结果表明,HbBAM1定位于叶绿体中;HbBAM1原核表达产物的最适酶活性温度是35℃,其酶活性受到氧化型谷胱甘肽和双氧水等氧化剂的抑制。HbBAM1基因主要在橡胶树的花和叶片中表达;HbBAM1基因随着叶片的发育进程表达量逐渐增加,在淡绿期和稳定期叶片中表达量最大;HbBAM1基因在成熟叶片中的表达呈现明显的昼夜差异,在光合作用最强的上午10:00和下午16:00的表达量最大,晚上的表达量最低。上述结果表明,HbBAM1可能参与橡胶树叶片叶绿体内淀粉的降解,控制叶片气孔的开放与关闭,从而调控橡胶树叶片的光合作用。      

香蕉NPR1E基因的克隆及表达分析

NPR1基因可参与调节植物对病原菌的广谱抗性,在植物系统抗性中起着关键的调控作用。通过RACE(rapid-amplification of cDNA ends)方法从香蕉根系中获得NPR1E基因,命名为MaNPR1E(GenBank登录号分别为：KF582550)。MaNPR1E是香蕉NPR1基因编码框全长cDNA,包含一个1 755 bp的最大开放阅读框,编码一个含584个氨基酸的蛋白质。经蛋白质序列同源比对发现,其含有完整的BTB/POZ结构域、锚蛋白重复ANK序列和NPR1-like-C结构域,属于典型的NPR1蛋白。系统进化树比对分析表明,MaNPR1E与海枣PdNPR3(XP_008808341.1)和油棕EgNPR3(XP_010914123.1)的亲缘关系较近。组织特异性研究表明,该基因组成型表达于香蕉各组织。实时荧光定量PCR分析表明,接种枯萎病菌后MaNPR1E的表达在感病品种中被抑制,而在抗病品种中被激活;MaNPR1E的表达受乙烯利和水杨酸的诱导。上述结果表明,MaNPR1E可能在香蕉抗枯萎病过程中扮演重要的调控角色。     

大豆GmNCED1基因的克隆及表达模式分析

本研究以抗逆性强的大豆旱碱一号为材料,首次从中克隆了编码9-顺式-环氧类胡萝卜素双加氧酶（9-cis-epoxycarotenoid dioxygenase,NCED）的GmNCED1基因全长cDNA片段,该基因编码区含有1 836个核苷酸,编码611个氨基酸。通过同源性比对分析发现GmNCED1与花生、菜豆等双子叶豆科植物NCED的同源性高达84%以上。同时,利用荧光定量PCR分析发现高盐、低温、干旱、外源ABA以及NAA处理均可以诱导该基因在大豆叶片及根中表达。本研究初步揭示GmNCED1基因在植物逆境胁迫中的作用,为基因工程育种提供优质的候选基因。